Recently, productivity of protein preparations typified by antibody preparations has been improved through mass fermentation and high fermentation titer. With this, it is desired to improve efficiency of the purification process. In particular, improvement of flow rate characteristics and adsorption characteristics of chromatography packing materials to be used for purification is desired because cost reduction is led thereby.
In order to realize higher flow rates, chromatography packing materials produced using a technique of crosslinking a base gel to increase its strength have been developed. Moreover, it is known that adsorption characteristics are improved by combining these crosslinked gels with a hydrophilic polymer such as dextran. Such hydrophilic polymer-introduced porous base materials are attracting attention in the area of chromatography packing material development.
For example, Non-Patent Document 1 (Journal of Chromatography A, 679 (1994) 11-22) reports that adsorption performance for proteins is improved by using an ion exchange adsorbent made of dextran-derived hydrogel in which an ion exchange group is added to core particles made of polystyrene-silica (commercially available as “S HyperD (trademark)” (Biosepra, France)).
Further, Patent Document 1 (Japanese Laid-Open Patent Publication No. 2008-528966) reports that adsorption characteristics for proteins were improved by using a chromatography packing material made by adding polyethylenimine to methacrylate polymer particles.
Moreover, Patent Document 2 (Japanese Laid-Open Patent Publication No. 2008-232764) describes that a strong-cation ion exchange chromatography matrix having good flow rate characteristics was successfully obtained by introducing a ligand into a gel in which pullulan having a molecular weight of 500,000 was immobilized to porous methacrylate particles through 2-bromoethanesulfone. It is reported that in this case, the adsorption capacity of human immunoglobulin was 160 mg/ml.
Non-Patent Document 2 (Journal of Chromatography A, 1146 (2007) 202-215) reports the effects exerted when the surface of core particles made of agarose was modified using dextran. It is reported that by modifying the surface of core particles using dextran, diffusion characteristics of a target protein in a carrier is improved, resulting in improvement of mass transfer of the target. As an example of a chromatography packing material designed as described above, “SP Sepharose (trademark) XL” can be obtained from GE Healthcare Sciences.
Furthermore, Patent Document 3 (International Publication WO 2007/027139 pamphlet) reports that, as an example of a cation exchange chromatography gel matrix that can be utilized for production of antibody preparations, a gel having immunoglobulin (IgG) adsorption performance of 143 mg/ml was successfully obtained by introducing an ion exchange group into particles made by allowing dextran having a molecular weight of 40 kDa to be covalently bound a crosslinked agarose gel using vinyl sulfonic acid.
As described above, it has been reported that adsorption performance is improved by adding a hydrophilic polymer to a base gel. However, particularly in the field of the production of antibody preparations, improvement of high capacity and high titer in the production has been diligently practiced. Therefore, it is desired to further improve high flow rate characteristics and dynamic adsorption characteristics of base materials for purification.
Packing materials comprising silica as a base material, which are typical chromatography packing materials, are excellent in high flow rate characteristics. However, since the quality of materials thereof is unstable under alkali conditions, such packing materials are disadvantageous to general alkali washing.
On the other hand, chromatography packing materials using polysaccharides as base materials have high alkali resistance. Further, the chromatography packing materials using polysaccharides as base materials have porous property suitable for separation and purification of the protein size, and are promising materials for separation and purification of protein or vaccine preparations.
When making a comparison between cellulose and agarose, which are typical polysaccharides, cellulose has a robuster hydrogen-bonding network, and therefore advantageous for providing a higher flow rate desired for chromatography packing materials. However, in the case of using cellulose as a base material, flow rate characteristics and adsorption characteristics suitable for separation and purification of immunoglobulin for protein preparations and in particular, antibody preparations have not been realized.